Genome-wide DNA Methylation Analysis Of Age-related Hearing Loss

Objective:Age-related hearing loss(ARHL),the most prevalent sensory impairment among middle-aged and elderly adults,has become a critical public health issue in the world.Studies has confirmed that ARHL is a complex disease involving environment and genetic susceptibility,which has negative impact on the physiological function,cognitive function and quality of life among middle-aged and elderly people.The occurrence and development of hearing loss may be related to DNA methylation,which is influenced by age,heredity and early living environment.The adopt of identical twin design can largely eliminate the mixed effects caused by genetic background and environmental factors.In order to investigate the role of DNA methylation in the occurrence and progression of ARHL,this study carried out genome-wide DNA methylation research on middle-aged and elderly identical monozygotic twins with different hearing phenotypes,and provided a reference for hearing epigenetics research.Methods:Sixty-one pairs of monozygotic twins were recruited through the Qingdao twin registration system,and field data were obtained by questionnaire surveys,pure tone audiometry,biological measurement and blood sample collection.Pure tone air conduction audiometer was used to measure the left and right ears at five different frequencies: 0.5k Hz,1.0k Hz,2.0k Hz,4.0k Hz,and 8.0k Hz.The Pure Tone Average(PTA)at the language frequencies(0.5k Hz,1.0k Hz,4.0k Hz)、low frequencies(0.5k Hz,1.0k Hz,2.0k Hz)and high frequencies(4.0k Hz,8.0k Hz)PTA4、PTA3、PTA2 and Better Ear Hearing Level(BEHL)at five frequencies,BEHL0.5、BEHL1、BEHL2、BEHL4 and BEHL8 were selected as the hearing phenotype.Sex,ABO blood type,and microsatellite DNA genotyping techniques were used to determine the zygosity.The DNA methylation profile was investigated using simplified reduced representative bisulfite sequencing technology,whereas the m RNA gene expression profile was investigated using second-generation sequencing technology.The linear mixed effect model was used to investigate the association between a single cytosinephosphate-guanosine(Cp G)site of DNA methylation level and hearing.Differentially Methylated Regions(DMRs)related to hearing were identified by the comb-p python software.The online genomic regions enrichment of annotations tool(GREAT)was used to investigate hearing-related cis-regulatory regions and biological pathways.Weighted gene correlation network analysis(WGCNA)was utilized to investigate the gene modules and genes associated with hearing using data from gene expression profiles.Results:1.The study included 61 pairs of monozygotic twins in all,the specific subjects were as follows: 57 pairs(PTA4),54 pairs(PTA3),54 pairs(PTA2),50 pairs(BEHL0.5),49 pairs(BEHL1),40 pairs(BEHL2),51 pairs(BEHL4)and 55 pairs(BEHL8).2.Based on the Epigenome-Wide Association Study of single Gp G,140 Cp Gs were found:42(PTA)、18(PTA3)、12(PTA2)、13(BEHL0.5)、23(BEHL1)、15(BEHL2)、13(BEHL4)、4(BEHL8).These loci were annotated on or near 36 genes,involving SPATA18,SPEF1,DUSP4,OSR1,SMAD9,LCK,C21orf58,LCK,TUSC1 and other genes.3.Based on the analysis of differential methylation regions,56 significant DMRs were identified: 6(PTA3),11(Speech-PTA),6(PTA2),11(BEHL0.5),10(BEHL1),4(BEHL2),5(BEHL4)and 3(BEHL8).These DMRs are located in or near 24 genes,among them,the methylation level of 29 DMRs is negatively correlated with hearing,11 DMRs are positively correlated,and the correlation of 16 DMRs is difficult to determine.4.GREAT analysis has enriched some pathways that may be related to hearing,including adrenaline and norepinephrine biosynthesis pathway,calcium transport pathway,endogenous cannabinoid pathway and so on.5.A total of 80 key hub genes were screened from four meaningful modules of WGCNA.Conclusion:Numerous Cp G sites,DMRs,pathways,and genes that may be connected to hearing were found in this study,which will offer some reference ideas and basis for other epigenetics research related to hearing in the future.